The invention relates to fusion protein expression systems for use in mammalian cells that enhance the production of a given target protein. More specifically, the invention relates to a secretion cassette, comprised of a mammalian signal peptide and a portion of mammalian immunoglobulins, which, when used as the amino-terminal fusion partner to the target protein, generally leads to high level expression and secretion of the fusion product. Such fusion proteins are useful, for example, for the production and extracellular collection of target proteins without the need for lysis of a host cell. The invention is perhaps most useful for the expression of target proteins which are not normally secreted from a host cell, are secreted at low levels from a host cell, or are toxic or otherwise deleterious to a host cell.
Expression systems employing gene fusion constructs have been used to enhance the production of proteins in bacteria. Employing a bacterial protein that is normally expressed at a very high level as the amino-terminal fusion partner of a fusion protein helps to ensure efficient transcription and translation of the message, and in some cases the secretion and solubilization of the fusion protein (Smith and Johnson (1988) Gene 67:31; Hopp et al. (1988) Biotechnology 6:1204; La Vallie et al. (1993) Biotechnology 11:187).
The major goal of expression of recombinant fusion proteins in mammalian cells has been to confer novel properties to the hybrid molecules, e.g., targeting of a cytokine or toxin in vivo, Fc receptor binding, complement fixation, protein A binding, increasing the half-life, and crossing the blood-brain barrier. Examples of recombinant fusion proteins produced in mammalian cells include cytokine immunoconjugates (Gillies et al. (1992) Proc. Natl. Acad. Sci. USA 89:1428; Gillies et al. (1993) Bioconjugate Chemistry 4:230), immunoadhesins (Capon et al. (1989) Nature 337:525), immunotoxins (Chaudhary et al. (1989) Nature 339:394), and a nerve growth factor conjugate (Friden et al. (1993) Science 259:373). Each of the foregoing publications is incorporated herein by reference. Proteins produced in mammalian cells often do not have the solubility and secretion problems encountered in bacterial expression. The use of gene fusion constructs to enhance the production or secretion of a target protein in a mammalian system has not been explored fully.
It is the object of the invention to provide DNAs which facilitate the production and secretion of a target protein. In particular, objects of the invention are to provide novel DNAs which: facilitate efficient production and secretion of hard to express proteins, such as nuclear proteins, regulatory proteins and proteins which otherwise may be toxic to a host cell, and can be adapted to any target polypeptide of interest which can be coded for and expressed in a host organism; to provide DNA constructs for the rapid and efficient production and secretion of proteins in a variety of host cells; and to provide a method for the production, secretion and collection of genetically engineered proteins, including non-native, biosynthetic, or otherwise artificial proteins, such as proteins which have been created by rational design. Other objects of the invention are to provide DNA sequences which, when fused to a polynucleotide encoding a target protein, encode a fusion polypeptide which can be purified using common reagents and techniques, and to interpose a proteolytic cleavage site between the encoded secretion cassette and the encoded target protein such that the secretion cassette can be cleaved from the target protein and the target protein can be purified independently. Still another object is to provide a procedure which is both efficient and inexpensive.
These and other objects of the invention will be apparent from the description, drawings, and claims that follow.